This invention relates to the development of a chemically stable formulation of plant defensive response elicitors and their use in agricultural and horticultural crops.
Plants have developed a number of defensive strategies against pathogens and herbivores. These include structural modifications such as leaf shape, thorns, and trichomes, as well as varied biochemical strategies developed by plants to deter insect and pathogen attacks. The innate ability of plants to resist insect feeding and pathogen infection is known but little understood. It has, however, been used as selection criteria by classical plant breeders in developing new resistant crop varieties. In recent years, using new technologies scientists have made great strides in understanding the mechanisms of plant defenses and the molecular components involved.
Plant Defense Responses
To date, four distinct but overlapping plant defensive responses have been identified. These are the hyper sensitive response (HR), the wounding response (WR), the systemic acquired resistance response (SAR), and the induced systemic resistance response (ISR) (1,2).
The hyper sensitive response (HR) is the first line of defense used by plants against pathogen infections and occurs rapidly after the detection of the pathogen at the site of infection. The characteristic feature of this mechanism is the development of lesions, whereby the plant cells surrounding the infection site die either thorough direct interaction with the pathogen or by an endogenous self-destruct mechanism to produce lesions, hence reducing the spread of the pathogen.
Wounding or insect feeding activates the Wound Response. In this response, the plant produces a number of proteins, including proteinase inhibitors. As the insect continues to feed on plant tissues, it will start to ingest the newly synthesized proteins. The function of the plant proteinase inhibitor proteins is to inhibit the insect""s digestive enzymes and hence deter its feeding.
Following the HR and or WR responses, the plant produces a number of proteins, collectively referred to as pathogenesis related (PR) proteins (3). The PR proteins are synthesized not only locally at the point of attack, but also systemically through out the plant. This second stage response is known as Systemic Acquired Resistance (SAR). The SAR response primes the whole plant to repel the pathogen or insect, not only at the original point but also throughout the plant.
The above three pathways are initiated by the plant in response to infection or insect feeding which could cause harm to the plant. The ISR response, however, is distinct because its activation is caused by non-pathogenic and beneficial rhizobacteria and the components of their cell walls. The ISR response acts to xe2x80x9cimmunizexe2x80x9d the plant against infection by pathogenic organisms (4, 5).
The ability of a plant to develop a systemic resistance after localized infection or wounding indicates that it possesses signaling mechanisms, which not only alerts cells close to the point of infection or injury but also cells distal to the interaction site.
Signaling Molecules Involved in the Plant Defensive Response
Unlike animals, plants do not possess a nervous system. Instead, plants have developed signal molecules to facilitate communication between cells. Three prominent signaling molecules used by plants also play major roles in initiating the defense response. These molecules are salicylic acid (SA), jasmonic acid (JA), and ethylene.
Salicylic acid (C7H6O3) is a phenolic compound produced by plants and is the key signal molecule for the initiation of the SAR response (6, 7) after pathogen infection (FIG. 1). The plant growth regulators jasmonic acid (C12H18O3) (8) and ethylene (9) have also been shown to play key roles in the plant defensive responses (9-14). Ethylene and JA appear to work synergistically to initiate the SA independent pathways (FIG. 1) (15, 16). Even though these signals are produced in response to different stimuli and initiate different plant responses (17), they have significant effect on one another""s modes of action. For example, it has been shown that induction of the SAR response by exogenous application of SA has a negative effect on the synthesis and mode of action of JA and ethylene (18, 19).
There is also sufficient evidence available to suggest the presence of other endogenous plant signaling molecules whose nature and mode of action is yet to be determined. The plant defense response, however, is not solely mediated by endogenous plant signals. Exogenous molecules, both synthetic (36) and those produced through the interaction of the invading organism with the plant, have also been shown to act as elicitors of the plant defense responses (20, 21). The biopolymer chitosan is one such molecule. Chitosan is a long chain polymer of glucosamine molecules linked together via xcex2-(1,4) linkages and is a cell wall component of a number of fungi (22, 23). Chitosan has been shown to have some direct anti-fungal and antibacterial activities (24, 25) and has been proposed for use as a food preservative.
Application of chitosan to plants causes a number of different physiological responses. It reduces the stomatal apertures of the leaf, thus reducing the ability of pathogens to gain access into the plant (26), causes the production of phenolic compounds (27, 28), and increases crop yields (29, 30). Chitosan also acts as an effective elicitor of the plant defense response and causes a decrease in disease incidence in treated crops (31-35).
Synergistic Effect of Salicylic Acid and Chitosan
Recently, the application of plant defensive elicitors has been recognized as a viable alternative to the use of more traditional pesticides, and new commercial products such as Messenger(copyright) and Actigard(copyright) have been developed (21, 36). Because the plant defense response is mediated by a number of signaling molecules that activate different plant defensive pathways, the external application of a single elicitor would under-utilize the full array of defensive responses available to the plant. In addition, the plant defense response is transient, i.e., the genes involved are switched on due to a stimulus, such as a pathogen or insect attack. After the stimulus is removed, the plant switches off the genes. Typically within six weeks after the removal of the stimulant (elicitor), the plant gradually degrades and reabsorbs the defensive gene products. Because the plant defense response is transient in nature, in order to utilize it as a viable method of protection it is necessary to constantly stimulate the plant.
After foliar or soil application, salicylic acid is readily taken up by plants and induces the SAR response. It is however, short-lived both in the plant and in the environment (37), which limits its effectiveness as an elicitor. In addition, in some plants salicylic acid""s induction of SAR causes a shut down of the JA-induced pathways (18, 19). Chitosan, on the other hand, is more stable and is gradually absorbed by the plant. It also has a broader range of activities, acting both on the salicylic acid-dependent and salicylic acid-independent pathways. It also mitigates some of the effects of salicylic acid on the other defensive pathways (18, 31). The elicitor effect of chitosan is dependent on the degree of polymerization of the molecules. Chitosan oligomers of between 2-50 monomers in length have been shown to be the most effective in eliciting the plant""s defensive responses.
The combinational use of SA and chitosan has a synergistic effect whereby SA allows for a rapid induction of the SAR response, which is then maintained and augmented through the slower action of the chitosan as it is gradually absorbed by the plant. Long chain chitosan polymers as well as short chain chitosan polymers are, however, practically in soluble at pH 7 or above. Thus, there is a need in the art for a method of preparing a formulation of chitosan and salicylic acid, which allows for the solubilization of all the components over a wide range of pH values.
It is an object of the invention to provide stable aqueous formulations of chitosan and salicylic acid. This and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a stable aqueous formulation comprising at least about 4% salicylic acid, hydrolyzed chitosan in a molar ratio with the salicylic acid ranging from about 0.01:1 to about 3:1, an inorganic base in a molar ratio with the salicylic acid of about 0.2:1 to about 3:1, and humic acid in a weight to weight ratio with the hydrolyzed chitosan ranging from about 1:1 to about 1:10.
Another embodiment of the invention is a stable aqueous formulation comprising at least about 2% hydrolyzed chitosan, salicylic acid in a molar ratio with the hydrolyzed chitosan ranging from about 1:0.01 to about 1:3, an inorganic base in a molar ratio with the salicylic acid of about 0.2:1 to about 3:1, and humic acid in a weight to weight ratio with the hydrolyzed chitosan ranging from about 1:1 to about 1:10.
Yet another embodiment of the invention is a stable aqueous formulation comprising at least about 4% salicylic acid, at least about 2% hydrolyzed chitosan, wherein the hydrolyzed chitosan is present in a molar ratio with the salicylic acid ranging from about 0.01:1 to about 3:1, an inorganic base in a molar ratio with the salicylic acid of about 0.2:1 to about 3:1, and humic acid in a weight to weight ratio with the hydrolyzed chitosan ranging from about 1:1 to about 1:10.
Even another embodiment of the invention is a stable aqueous formulation comprising at least about 4% salicylic acid, hydrolyzed chitosan in a molar ratio with the salicylic acid of about 1:1, an inorganic base in a molar ratio with the salicylic acid of about 1:1, and humic acid in a weight to weight ratio with the hydrolyzed chitosan of about 1:7.
A further embodiment of the invention is a stable aqueous formulation comprising at least about 4% salicylic acid, hydrolyzed chitosan in a molar ratio with the salicylic acid ranging from about 0.01:1 to about 3:1, an inorganic base in an amount sufficient to stabilize the salicylic acid, and humic acid in an amount sufficient to stabilize the hydrolyzed chitosan.
Another embodiment of the invention is a stable aqueous formulation comprising at least about 2% hydrolyzed chitosan, salicylic acid in a molar ratio with the hydrolyzed chitosan ranging from about 1:0.01 to about 1:3, an inorganic base in an amount sufficient to stabilize the salicylic acid, and humic acid in an amount sufficient to stabilize the hydrolyzed chitosan.
Still another embodiment of the invention is a method of making a stable aqueous formulation of salicylic acid and chitosan. Hydrolyzed chitosan is added to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 4% salicylic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10.
Yet another embodiment of the invention is a method of making a stable aqueous formulation of salicylic acid and chitosan. Hydrolyzed chitosan is added to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 2% humic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10.
Another embodiment of the invention is a stable aqueous formulation made by adding hydrolyzed chitosan to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 4% salicylic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10.
Yet another embodiment of the invention is a stable aqueous formulation made by adding hydrolyzed chitosan to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 2% humic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10.
Still another embodiment of the invention is a stable aqueous formulation comprising about 31% salicylic acid, about 16% hydrolyzed chitosan, about 11% potassium hydroxide, and about 2% humic acid.
A further embodiment of the invention is a method of making a stable aqueous formulation of salicylic acid and hydrolyzed chitosan. Hydrolyzed chitosan is added to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 31% salicylic acid and at least about 16% chitosan.
Another embodiment of the invention is a stable aqueous formulation made by adding hydrolyzed chitosan to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 31% salicylic acid and at least about 16% chitosan.
Yet another embodiment of the invention is a method of enhancing a plant defensive response against a pathogen or herbivore. A plant is contacted with a formulation comprising about 5% by weight of a stable formulation made by adding hydrolyzed chitosan to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 4% salicylic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10. The plant""s defensive response is thereby enhanced.
Yet another embodiment of the invention is a method of enhancing a plant defensive response against a pathogen or herbivore. A plant is contacted with a formulation comprising about 5% by weight of a stable formulation made by adding hydrolyzed chitosan to a first aqueous solution to form a second aqueous solution. The first aqueous solution comprises salicylic acid and an inorganic base in an amount sufficient to stabilize the salicylic acid. Humic acid is added to the second solution in an amount sufficient to stabilize the hydrolyzed chitosan. The stable formulation comprises at least about 2% humic acid. The salicylic acid and the hydrolyzed chitosan are present in a molar ratio ranging from about 1:0.01 to about 1:3. The inorganic base and the salicylic acid are present in a molar ratio of about 0.2:1 to about 3:1. The humic acid and the hydrolyzed chitosan are present in a weight to weight ratio ranging from about 1:1 to about 1:10. The plant""s defensive response is thereby enhanced.
The invention thus provides stable, aqueous formulations comprising chitosan and salicylic acid, as well as methods of making such formulations and using them to enhance plant defensive responses.